Conditional antagonism in co-cultures ofPseudomonas aeruginosaandCandida albicans: an intersection of ethanol and phosphate signaling distilled from dual-seq transcriptomics

Abstract

1Pseudomonas aeruginosa and Candida albicans are opportunistic pathogens 2 whose interactions involve the secreted products ethanol and phenazines. Here we 3 describe the focal role of ethanol in mixed-species co-cultures by dual RNA-seq analyses. 4 P. aeruginosa and C. albicans transcriptomes were assessed after growth in mono-5 culture or co-culture with either ethanol-producing C. albicans or a C. albicans mutant 6 lacking the primary ethanol dehydrogenase, Adh1. Analyses using KEGG-pathways and 7 the previously published eADAGE method revealed several P. aeruginosa responses to 8 C. albicans-produced ethanol including the induction of a non-canonical low phosphate 9 response mediated by PhoB. C. albicans wild-type, but not C. albicans adh1∆/∆, induces 10 P. aeruginosa production of 5-methyl-phenazine-1-carboxylic acid (5-MPCA), which 11 forms a red derivative within fungal cells. We first demonstrate that PhoB is required for 12 this interaction and that PhoB hyperactivity, via deletion of pstB, leads to increased 13 production of 5-MPCA even when phosphate concentrations are high, but only in the 14 presence of ethanol. Second, we show that ethanol is only sufficient to promote 5-MPCA 15 production at permissive phosphate concentrations. The intersection of ethanol and 16 phosphate in co-culture is mirrored in C. albicans; the adh1∆/∆ mutant had increased 17 expression of genes regulated by Pho4, the C. albicans transcription factor that responds 18 to low phosphate which we confirmed by showing the adh1∆/∆ strain had elevated Pho4-19 dependent phosphatase activity. The dual-dependence on ethanol and phosphate 20 concentrations for anti-fungal production highlights how environmental factors modulate 21 microbial interactions and dictate antagonisms such as those between P. aeruginosa and 22 C. albicans. 23 24Author Summary 25 26 Pseudomonas aeruginosa and Candida albicans are opportunistic pathogens that are 27 frequently isolated from co-infections. Using a Dual-Seq approach in combination with 28 genetics approaches, we found that ethanol produced by C. albicans stimulates the PhoB 29 regulon in P. aeruginosa asynchronously with activation of the Pho4 regulon in C. 30 albicans. In doing so, we demonstrate that eADAGE-based analysis can improve the 31 understanding of the P. aeruginosa response to ethanol-producing C. albicans as 32 measured by transcriptomics: we identify a subset of PhoB-regulated genes as 33 differentially expressed in response to ethanol. We validate our result by showing that 34PhoB is necessary for multiple roles in co-culture including the competition for phosphate 35 and the production of 5-methyl-phenazine-1-carboxylic acid, and that the P. aeruginosa 36 response to C. albicans-produced ethanol depends on phosphate availability. The 37 conditional stimulation of virulence production in response to sub-inhibitory 38 concentrations of ethanol only under phosphate limitation highlights the importance of 39 considering nutrient concentrations in the analysis of co-culture interactions....

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